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1.  The loss of renal dendritic cells and activation of host adaptive immunity are long-term effects of ischemia/reperfusion injury following syngenic kidney transplantation 
Kidney International  2012;81(10):1015-1025.
Ischemia/reperfusion injury associated with kidney transplantation induces profound acute injury, influences early graft function and affects long-term graft outcomes. To determine whether renal dendritic cells play any role during initial innate ischemia/reperfusion injury and the subsequent development of adaptive immune responses, we studied the behavior and function of renal graft and host infiltrating dendritic cells during early and late phases of renal ischemia/reperfusion injury. Wild type to GFP-transgenic rat kidney transplantation was performed with and without 24 hours cold storage. Ischemia/reperfusion injury in cold stored grafts resulted in histopathological changes of interstitial fibrosis and tubular atrophy by 10 weeks accompanied by upregulation of mRNAs of mediators of interstitial fibrosis and inflammation. In normal rat kidneys we identified two populations of renal dendritic cells, predominant CD103−CD11b/c+ and minor CD103+CD11b/c+ cells. After transplantation without cold storage, grafts maintained CD103− but not CD103+ GFP-negative renal dendritic cells for 10 weeks. In contrast, both cell subsets disappeared from cold stored grafts, which associated with a significant GFP-expressing host CD11b/c+ cell infiltration that included CD103+ dendritic cells with a TNF-α producing phenotype. These changes in graft/host dendritic cell populations were associated with progressive infiltration of host CD4+ T cells with effector/effector-memory phenotypes and IFN-γ secretion. Thus, renal graft ischemia/reperfusion injury causes graft dendritic cell loss and was associated with progressive host dendritic cell and T cell recruitment. Renal resident dendritic cells might function as a protective regulatory network.
PMCID: PMC3340432  PMID: 22278023
CD103; IF/TA; effector CD4+ T cells; IFN-α; TNF-α
2.  99mTc-MAG3 scintigraphy for the longitudinal follow-up of kidney function in a mouse model of renal ischemia-reperfusion injury 
EJNMMI Research  2012;2:2.
Experimental models are essential tools in the development and evaluation of novel treatment options, but the preclinical model of renal ischemia-reperfusion injury is limited to the retrieval of (very) early functional data, leaving the pivotal long-term outcome unknown. The present study applies technetium-99m-mercapto-acetyl-tri-glycine [99mTc-MAG3] scintigraphy for the longitudinal follow-up examination of long-term kidney function after renal ischemia-reperfusion injury.
Unilateral warm ischemia was induced in scid beige mice by vascular clamping of the kidney hilum for 40 min. 99mTc-MAG3 scintigraphy was performed prior to injury, 8 and 14 days post ischemia. The fractional uptake rate [FUR] was calculated from scintigraphy data as a measure of renal clearance.
FUR demonstrated a significant functional impairment of the ischemic kidney 8 and 14 days after injury (P < 0.05 vs. baseline), while contralateral non-ischemic kidneys showed no significant changes. In histological analysis, ischemic kidneys exhibited tubular dilatation and cytoplasmic degeneration as signs of hypoxia without any evidence for necrosis.
FUR enables the detection of renal dysfunction and longitudinal long-term follow-up examination in the same individual. Our model may facilitate preclinical therapy evaluation for the identification of effective renoprotective therapies.
PMCID: PMC3282629  PMID: 22264389
fractional uptake rate; ischemia-reperfusion injury; 99mTc-MAG3 scintigraphy; renal clearance; tubular function
3.  Reduced Lung Function in a Chronic Asthma Model Is Associated with Prolonged Inflammation, but Independent of Peribronchial Fibrosis 
PLoS ONE  2008;3(2):e1575.
In asthma, mechanisms contributing to chronicity remain to be determined. Recent models of sensitisation with prolonged airway allergen challenges reproduce typical features of chronic asthma. However, the interplay between inflammation, structural changes and lung function is poorly understood. This study was performed to delineate functional, structural and immunological airway changes after cessation of long term challenges to elucidate factors contributing to the development of prolonged lung function changes.
Methodology/Principal Findings
Mice sensitised systemically were consecutively challenged intranasally with ovalbumin for two or eight weeks. After the end of challenges, lung function, airway inflammation, features of airway remodelling, local T-cell cytokines and systemic ovalbumin-specific antibodies were monitored. Long term challenges resulted in airway hyperresponsiveness lasting 2 weeks and reduced baseline lung function for 6 weeks after their cessation. In contrast, these changes resolved within one week after short term challenges. Prolonged transforming growth factor beta (TGF-β)1 production and marked peribronchial fibrosis were only induced by long term challenges. Importantly, fibrosis became apparent only after the onset of lung function changes and outlasted them. Further, long term challenges led to prolonged and intense airway inflammation with marked lymphocytosis, but moderate eosinophilia, sustained IL-5 production and ovalbumin-specific IgG2a antibodies, the latter suggesting a Th1 component to the immune response. In contrast, following short term challenges airway inflammation was dominated by eosinophils and associated with a strong, but transient IL-13 response.
Prolonged lung function changes after long term allergen challenges seem to develop and resolve independently of the persistent peribronchial fibrosis. They are more closely associated with intense airway inflammation, marked lymphocytosis, prolonged IL-5 and TGF-β1 production in the airways and a Th1 immune response.
PMCID: PMC2215329  PMID: 18253511
4.  Primary Graft Dysfunction 
Primary graft dysfunction (PGD) is a syndrome encompassing a spectrum of mild to severe lung injury that occurs within the first 72 hours after lung transplantation. PGD is characterized by pulmonary edema with diffuse alveolar damage that manifests clinically as progressive hypoxemia with radiographic pulmonary infiltrates. In recent years, new knowledge has been generated on risks and mechanisms of PGD. Following ischemia and reperfusion, inflammatory and immunological injury-repair responses appear to be key controlling mechanisms. In addition, PGD has significant impact on short- and long-term outcomes; therefore, the choice of donor organ is impacted by this potential adverse consequence. Improved methods of reducing PGD risk and efforts to safely expand the pool are being developed. Ex-vivo lung perfusion is a strategy which may improve risk assessment and become a promising platform to implement treatment interventions to prevent PGD. This review will detail recent updates in the epidemiology, pathophysiology, molecular and genetic biomarkers and state-of-the-art technical developments affecting PGD. (158 words)
PMCID: PMC3968688  PMID: 23821506
primary graft dysfunction; lung transplantation; ischemia-reperfusion injury and repair; high-risk donor lung; ex-vivo lung perfusion
Surgery  2009;146(2):181-189.
Liver ischemia and reperfusion (IR) injury is a phenomenon that leads to graft dysfunction following liver transplantation. Understanding the molecular mechanisms behind this process is crucial to developing strategies to prevent short and long term graft dysfunction. The purpose of this study is to explore the role of the transcription factor, IRF-1, in a model of orthotopic rat liver transplantation.
Orthotopic syngeneic LEW rat liver transplantation (OLT) was performed after 18 or 3 hours preservation in cold UW solution. AdIRF-1 or control gene vector (Adnull) was delivered to the liver by donor intravenous pretreatment 4 days before graft harvesting. Uninfected grafts also served as controls. Recipients were sacrificed 1 to 24 hours post-transplantation.
Rats that underwent OLT with long-term preserved graft (18 hours) displayed increased hepatic nuclear expression of IRF-1 protein at 1 and 3 hours. Rats pre-treated with AdIRF-1 prior to transplantation had increased ALT levels and increased expression of IFN-β, IFN-γ, IL-12, and iNOS in short-term period graft(3 hours) when compared with donor livers pre-treated with Adnull. AdIRF-1 pre-treated donor livers also exhibited increased susceptibility to early apoptosis in the transplanted grafts with increased TUNEL staining expression of cleaved caspase-3. Additionally, AdIRF-1 pre-treated donor livers had increased activation of the MAP kinase JNK as compared with Adnull pre-treated donor livers.
IRF-1 is an important regulator of IR injury after OLT in rats. Targeting of IRF-1 may be a potential strategy to ameliorate ischemic liver injury after transplantation in order to minimize organ dysfunction.
PMCID: PMC2739669  PMID: 19628072
6.  Effect of Valproic Acid on Acute Lung Injury in a Rodent Model of Intestinal Ischemia Reperfusion 
Resuscitation  2011;83(2):243-248.
Acute lung injury (ALI) is developed in many clinical situations and associated with significant morbidity and mortality. Valproic acid (VPA), a well-known anti-epileptic drug, has been shown to have anti-oxidant and anti-inflammatory effects in various ischemia/reperfusion (I/R) models. The purpose of this study was to investigate whether VPA could affect survival and development of ALI in a rat model of intestinal I/R.
Two experiments were performed. Experiment I: Male Sprague-Dawley rats (250–300 g) were subjected to intestinal ischemia (1 hour) and reperfusion (3 hours). They were randomized into 2 groups (n=7/group) 30 min after ischemia: Vehicle (Veh) and VPA (300 mg/kg, IV). Primary end-point for this study was survival over 4 hours from the start of ischemia. Experiment II: The histological and biochemical effects of VPA treatment on lungs were examined 3 hours (1 hr ischemia + 2 hrs reperfusion) after intestinal I/R injury (Veh vs. VPA, n = 9/group). An objective histological score was used to grade the degree of ALI. Enzyme linked immunosorbent assay (ELISA) was performed to measure serum levels of cytokine interleukins (IL-6 and 10), and lung tissue of cytokine-induced neutrophil chemoattractant (CINC) and myeloperoxidase (MPO). In addition, the activity of 8-isoprostane was analyzed for pulmonary oxidative damage.
In Experiment I, four-hour survival rate was significantly higher in VPA treated animals compared to Veh animals (71.4% vs. 14.3%, p = 0.006). In Experiment II, ALI was apparent in all of the Veh group animals. Treatment with VPA prevented the development of ALI, with a reduction in the histological score (3.4 ± 0.3 vs. 5.3 ± 0.6, p = 0.025). Moreover, compared to the Veh control group the animals from the VPA group displayed decreased serum levels of IL-6 (952 ± 213 vs. 7709 ± 1990 pg/ml, p = 0.011), and lung tissue concentrations of CINC (1188 ± 28 vs. 1298 ± 27, p < 0.05), MPO activity (368 ± 23 vs. 490 ± 29, p <0.05) and 8-isoprostane levels (1495 ± 221 vs. 2191 ± 177 pg/ml, p < 0.05).
VPA treatment improves survival and attenuates ALI in a rat model of intestinal I/R injury, at least in part, through its anti-oxidant and anti-inflammatory effects.
PMCID: PMC3242830  PMID: 21824465
valproic acid; acute lung injury; ischemia reperfusion; inflammation; oxidative damage; intestine
7.  Role of Ethyl Pyruvate in Systemic Inflammatory Response and Lung Injury in an Experimental Model of Ruptured Abdominal Aortic Aneurysm 
BioMed Research International  2014;2014:857109.
Objectıve. The purpose of this study is to evaluate the effect of ethyl pyruvate (EP) on systemic inflammatory response and lung injury in an experimental rat model of ruptured abdominal aortic anurysm (RAAA). Methods. Anaesthetized 30 Sprague-Dawley male rats were randomized to sham (Sh n : 6) (Sh + EP n : 6) or shock and clamp (S/C) groups (S/C n : 9) (S/C + EP n : 9). In the S/C and S/C + EP groups, hemorrhagic shock, lower torso ischemia, and reperfusion were created, S/C group was given 1 mL saline and S/C + EP group was given 40 mg/kg EP. At the end of reperfusion process some biochemical and histological parameters were studied in serum and lung tissues. Results. An increase was observed in all parameters except interleukin-6 (IL-6) in the S/C group in comparison to the sham groups. In the S/C + EP group, serum myeloperoxydase (MPO), malondialdehyde (MDA), and tumor necrosis factor alpha (TNF-α) as well as lung MPO and MDA values decreased significantly (P < 0.016). In the lung tissues, histological injury scores and lung tissue wet/dry ratio were significantly decreased in the S/C + EP group as compared to the S/C group (P < 0.016). Conclusions. Ethyl pyruvate may reduce systemic inflammatory response and lung injury which resulted from shock and ischemia/reperfusion in an experimental model of RAAA.
PMCID: PMC3915710  PMID: 24575415
8.  Taurine protects against lung damage following limb ischemia reperfusion in the rat by attenuating endoplasmic reticulum stress-induced apoptosis 
Acta Orthopaedica  2010;81(2):263-267.
Background and purpose CHOP is a C/EBP family transcription factor involved in endoplasmic reticulum (ER) stress-mediated apoptosis. Several studies have demonstrated that ischemia reperfusion results in apoptosis. Oxidative stress is central to ischemia reperfusion-induced apoptosis. Taurine protects against lung injury after limb ischemia reperfusion (LIR) through antioxidation. The effects of taurine on ER stress-induced apoptosis have not been well explored, however. We studied the effects of taurine in ER stress-induced apoptosis following LIR.
Methods Adult male Sprague-Dawley rats (n = 40) were randomized into 4 groups: (1) a control group, (2) an LIR group, (3) an LIR group treated with taurine, and (4) an LIR group treated with saline. Bilateral hindlimb ischemia was induced by application of a rubber band proximal to the level of the greater trochanters for 4 h. The treatment groups received either taurine (200 mg/kg as a 4% solution in 0.9% saline) or saline alone prior to reperfusion. Following 4h of reperfusion, blood oxygen was analyzed. The animals were killed and plasma and lung tissue were harvested for evaluation.
Results Taurine statistically significantly attenuated lung injury following LIR, as shown by reduced malondialdehyde content, reduced cell apoptosis, and expression of activating transcription factor 4 (ATF4), X-box binding protein 1 (XBP1), and transcriptional activators of the CHOP gene. Furthermore, partial pressure values of oxygen in arterial blood and the activities of superoxide dismutase and catalase were higher in the taurine pretreatment group than in the group of rats that underwent LIR alone.
Interpretation Our results suggest that taurine attenuates endoplasmic reticulum stress-induced apoptosis in the lungs of rats after limb ischemia reperfusion.
PMCID: PMC2895349  PMID: 20148646
9.  A monoclonal antibody to the adherence-promoting leukocyte glycoprotein, CD18, reduces organ injury and improves survival from hemorrhagic shock and resuscitation in rabbits. 
Journal of Clinical Investigation  1988;81(3):939-944.
Leukocytes have been shown to play an important role in the development of isolated organ injury after experimental ischemia and reperfusion. To examine the role of leukocytes in generalized ischemia-reperfusion injury we used the MAb 60.3 (directed to the human leukocyte adherence glycoprotein, CD18) to block leukocyte adherence functions in a rabbit model of hemorrhagic shock and resuscitation. In control animals subjected to 1 h of shock (mean blood pressure 45 torr and mean cardiac output 30% of baseline) followed by resuscitation, only 29% survived 5 d. All had gross and histologic evidence of injury to lungs, liver, and gastrointestinal mucosa. In contrast, 100% of the MAb 60.3-treated animals survived 5 d (P less than 0.01) and organ injury was absent or markedly attenuated. The control animals also had a persistent acidosis, lost more weight, and had evidence of continued gastrointestinal bleeding in contrast to MAb 60.3-treated animals. We conclude that increased leukocyte adhesiveness plays an important role in the development of multiple organ injury and death after generalized ischemia-reperfusion and that this injury may be significantly reduced by blocking leukocyte adherence functions with the MAb 60.3.
PMCID: PMC442549  PMID: 3278007
10.  Effect of dexmedetomidine on lung ischemia-reperfusion injury 
Molecular Medicine Reports  2013;9(2):419-426.
Dexmedetomidine, a specific selective α2-adrenergic agonist, does not only have the characteristics of being a sedative and analgesic, but also exhibits a protective role in brain ischemia-reperfusion injury and inhibits the inflammation in animals with sepsis. The objective of the present study was to investigate whether dexmedetomidine is capable of attenuating rat pulmonary damage induced by ischemia-reperfusion injury, which is a type of acute sterile lung injury. Sprague-Dawley rats were randomly assigned into six groups: The sham-operated (sham) group, the lung ischemia-reperfusion (I/R) group, intravenous injection of dexmedetomidine 2.5 μg/kg/h (Dex2.5) or 5 μg/kg/h (Dex5) for 1 h prior to ischemia, combination of α2-adrenergic antagonist yohimbine prior to dexmedetomidine pre-treatment (Dex+Yoh) and pre-administration of yohimbine alone (Yoh) prior to ischemia. Lung injury was assessed by the histopathological changes, arterial blood gas, wet/dry (w/d) weight ratio and myeloperoxidase (MPO) activity of the lung. The concentration of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) in bronchoalveolar lavage fluid (BALF) was measured by an enzyme-linked immunosorbent assay. The expression of toll-like receptor-4 (TLR4) and myeloid differentiation factor 88 (MyD88) mRNA in the lung were determined by quantitative PCR, and phosphorylated levels of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK)1/2 were determined by western blotting. Pre-treatment with dexmedetomidine significantly reduced the lung injury, w/d weight ratio and MPO activity, and decreased the concentration of TNF-α, IL-6 and MCP-1 in BALF compared with the I/R group. The expression of TLR4 and MyD88 mRNA and the levels of phosphorylated JNK and ERK1/2 in the lung tissue were markedly downregulated by intravenous injection of dexmedetomidne for 1 h prior to lung I/R. The protective effects of dexmedetomidine on the lung were not completely reversed by the α2-adrenergic antagonist, yohimbine. Pre-treatment with dexmedetomidine is capable of reducing pulmonary damage and inhibiting sterile inflammation induced by lung I/R injury. TLR4/MyD88/mitogen-activated protein kinase (MAPK) signaling is involved in the protective mechanism of dexmedetomidine through α2-adrenoceptor independence.
PMCID: PMC3896524  PMID: 24345905
dexmedetomidine; ischemia-reperfusion injury; interleukin-6; tumor necrosis factor-α
11.  Lipopolysaccharide pre-conditioning is protective in lung ischemia-reperfusion injury 
The availability of suitable lung donors has remained a significant barrier to lung transplantation. The clinical relevance of an isolated positive Gram stain in potential donor lungs, which occurs in >80%, is unclear. Low doses of lipopolysaccharide (LPS) have been protective in several models of ischemia-reperfusion injury through a pre-conditioning response. We sought to demonstrate that low-dose LPS is protective against subsequent lung ischemia-reperfusion injury.
Pathogen-free Long-Evans rats were pre-treated with vehicle or LPS 24 hours before 90 minutes of ischemia and up to 4 hours of reperfusion. Lungs were assessed for vascular permeability, myeloperoxidase content, bronchoalveolar lavage inflammatory cell and cytokine/chemokine content, as well as nuclear translocation of nuclear factor κB (NFκB) and activator protein-1 (AP-1), and interleukin-1 receptor-associated kinase-1 (IRAK-1) and stress-activated protein kinase (SAPK) activation.
Compared with positive controls, LPS pre-treatment resulted in reductions in vascular permeability (70%, p < 0.001), myeloperoxidase content (93%, p < 0.001), bronchoalveolar lavage inflammatory cells (91%, p < 0.001), and inflammatory cytokine/chemokine content (cytokine-induced neutrophil chemoattractant, 99%, p = 0.003; interleukin-1β, 72%, p. 0.0001; tumor necrosis factor-α, 76%, p < 0.0001), NFκB (86%, p < 0.001) and AP-1 (97%, p < 0.001) nuclear translocation, and IRAK-1 (87%, p < 0.001) and SAPK (80%, p < 0.001) phosphorylation.
Lipopolysaccharide pre-treatment reduced lung injury and inflammatory mediator production after subsequent exposure to ischemia-reperfusion. Understanding the clinical significance of lipopolysaccharide in donor lungs has the potential to expand and clarify donor inclusion criteria.
PMCID: PMC3052281  PMID: 20044277
12.  Pharmacokinetics of ligustrazine ethosome patch in rats and anti-myocardial ischemia and anti-ischemic reperfusion injury effect 
The objective of this study was to investigate the pharmacokinetics of the ligustrazine ethosome patch and antimyocardial ischemia and anti-ischemic reperfusion injury effect. Male Sprague Dawley rats were divided randomly into 3 groups: Group A (intragastric ligustrazine), Group B (transdermal ligustrazine ethosome patch), and Group C (conventional transdermal ligustrazine patch). After treatment, samples of blood and of various tissues such as heart, liver, spleen, lung, kidney, brain, and muscle samples were taken at different time points. Drug concentration was measured with HPLC, and the drug concentration–time curve was plotted. Pharmacokinetic software 3p97 was applied to calculate pharmacokinetic parameters and the area under the drug concentration–time curve (AUC) in various tissues. The rat model of acute myocardial ischemia was constructed with intravenous injection of pituitrin and the model of myocardial ischemia-perfusion injury was constructed by tying off the left anterior descending coronary artery of rats to observe the effect of ligustrazine ethosome patches on ischemic myocardium and ischemia-reperfusion injury. Results showed that AUC was highest in the transdermal drug delivery group of ligustrazine ethosome patch. There were significant differences in whole blood viscosity, plasma viscosity, hematocrit, red blood cell aggregation index, and deformation index between ligustrazine the ethosome patch group and ischemic control group (P < 0.01). Moreover, ligustrazine ethosome patches could reduce the scope of myocardial infarction induced by long-term ischemia. Ligustrazine ethosome patches have a sustained-release property. They can maintain stable and sustained blood drug concentration, increase bioavailability, and reduce administration times. The drug patch can decrease hemorheological indices of myocardial ischemia in rats, as well as protect acute ischemic myocardium and ischemia-reperfusion injured myocardium.
PMCID: PMC3133529  PMID: 21760733
ligustrazine; ethosome; patch; pharmacokinetics; myocardial ischemia; ischemia- reperfusion injury
13.  Long-Term (Postnatal Day 70) Outcome and Safety of Intratracheal Transplantation of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells in Neonatal Hyperoxic Lung Injury 
Yonsei Medical Journal  2013;54(2):416-424.
This study was performed to evaluate the long-term effects and safety of intratracheal (IT) transplantation of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) in neonatal hyperoxic lung injury at postnatal day (P)70 in a rat model.
Materials and Methods
Newborn Sprague Dawley rat pups were subjected to 14 days of hyperoxia (90% oxygen) within 10 hours after birth and allowed to recover at room air until sacrificed at P70. In the transplantation groups, hUCB-MSCs (5×105) were administered intratracheally at P5. At P70, various organs including the heart, lung, liver, and spleen were histologically examined, and the harvested lungs were assessed for morphometric analyses of alveolarization. ED-1, von Willebrand factor, and human-specific nuclear mitotic apparatus protein (NuMA) staining in the lungs and the hematologic profile of blood were evaluated.
Impaired alveolar and vascular growth, which evidenced by an increased mean linear intercept and decreased amount of von Willebrand factor, respectively, and the hyperoxia-induced inflammatory responses, as evidenced by inflammatory foci and ED-1 positive alveolar macrophages, were attenuated in the P70 rat lungs by IT transplantation of hUCB-MSCs. Although rare, donor cells with human specific NuMA staining were persistently present in the P70 rat lungs. There were no gross or microscopic abnormal findings in the heart, liver, or spleen, related to the MSCs transplantation.
The protective and beneficial effects of IT transplantation of hUCB-MSCs in neonatal hyperoxic lung injuries were sustained for a prolonged recovery period without any long-term adverse effects up to P70.
PMCID: PMC3575965  PMID: 23364976
Stem cells; cell transplantation; animal model; newborn; inflammation
14.  Long-term evaluation of granulocyte-colony stimulating factor on hypoxic-ischemic brain damage in infant rats 
Intensive care medicine  2010;36(9):1602-1608.
Hypoxia-ischemia (HI), as a major cause of fetal brain damage, has long-lasting neurological implications. Therefore, therapeutic interventions that attenuate the neuropathological outcome of HI while also improving the neuro-functional outcome are of paramount clinical importance. The aim of this study was to investigate the long-term functional and protective actions of granulocyte-colony stimulating factor (G-CSF) treatment in an experimental model of cerebral HI.
Postnatal day-7 Sprague-Dawley rats were subjected to HI surgery, which entailed ligation of the right common carotid artery followed by 2 h of hypoxia (8% O2). Treatment consisted of subcutaneous injection of G-CSF at 1 h after hypoxia followed by an additional one injection per day for 5 days (6 total injections) or for 10 days (11 total injections). Animals were euthanized 5 weeks post-insult for extensive evaluation of neurological deficits and assessment of brain, spleen, heart, and liver damage.
G-CSF treatment promoted somatic growth and prevented brain atrophy and under-development of the heart. Moreover, reflexes, limb placing, muscle strength, motor coordination, short-term memory, and exploratory behavior were all significantly improved by both G-CSF dosing regimens.
Long-term neuroprotection afforded by G-CSF in both morphological and functional parameters after a hypoxic-ischemic event in the neonate provides a rationale for exploring clinical translation.
PMCID: PMC3133964  PMID: 20461500
G-CSF; Neurobehavior; Neonatal; Neuroprotection; Stroke; Hypoxia-ischemia
15.  Lung Injury after In Vivo Reperfusion 
Anesthesiology  2008;109(2):269-278.
Although short-term findings after lung reperfusion have been extensively reported, in vivo animal studies have not described outcome beyond the immediate time period. Therefore, the authors evaluated lung injury 27 h after reperfusion. They also investigated whether attenuation of lung injury with the A3 adenosine receptor agonist MRS3558 was sustained beyond the immediate time period.
In intact-chest, spontaneously breathing cats in which the left lower lung lobe was isolated and subjected to 2 h of ischemia and 3 h of reperfusion, MRS3558 was administered before reperfusion. Animals were killed 3 or 27 h after reperfusion.
When compared with 3 h of reperfusion, at 27 h the left lower lobe showed reduced apoptosis and no change in inflammation, but increased edema. Increased edema of the nonischemic right lung and hypoxemia were observed at 27 h after left lower lobe reperfusion. Increases in phosphorylated p38 levels were found at 3 h of reperfusion compared with control lung, with further increases at 27 h. The attenuation of injury observed with MRS3558 treatment at 3 h of reperfusion was sustained at 27 h.
Lung edema may worsen hours after the immediate postreperfusion period, even though lung apoptosis and inflammation are reduced or show no change, respectively. This was associated with further increases in phosphorylated p38 levels. The nonischemic lung may also be affected, suggesting a systemic response to reperfusion. In addition, early attenuation of injury is beneficial beyond the immediate period after reperfusion. Treatment aimed at inhibiting p38 activation, such as A3 receptor activation, should be further studied to explore its potential long-term beneficial effect.
PMCID: PMC2688693  PMID: 18648236
16.  Short- and long-term adverse effects of cocaine abuse during pregnancy on the heart development 
The effect of cocaine on the developing fetus is a topic of considerable interest and debate. One of the potential effects of fetal cocaine exposure is damage to the developing heart. This review provides an overview of the current understanding of the short- and long-term effects of fetal cocaine exposure on the heart in both humans and animal models. Human studies are still preliminary but have suggested that fetal cocaine exposure impacts on the developing heart. Studies in animal models provide strong evidence for a programming effect resulting in detrimental long-term changes to the heart induced by fetal cocaine exposure. In the rat model, fetal cocaine results in apoptosis in the term heart, left ventricular remodeling and myocyte hypertrophy, as well as increased sensitivity to ischemia/reperfusion injury in the adult male offspring. The rat model has also shown evidence of epigenetic modifications in response to intrauterine cocaine. Increased DNA methylation of promoter regions leads to a long-term decrease in the expression of the cardioprotective gene, PKCε. The current data shows fetal cocaine exposure has significant immediate and long-term cardiac consequences in animal models and while human studies are still incomplete they suggest this phenomenon may also be significant in humans exposed to cocaine during development.
PMCID: PMC2710813  PMID: 19144667
fetal; programming; cocaine
17.  Subcellular mechanisms of adaptation in the diabetic myocardium: Relevance to ischemic preconditioning in the nondiseased heart 
Although hyperglycemia is one factor that determines the outcome of myocardial ischemic insult, it is still not clear whether it is causally related to decreased ischemic tolerance in diabetic patients. In contrast to clinical and epidemiological studies demonstrating a higher risk of cardiovascular disorders in diabetic patients, experimental data are not unequivocal and suggest that, aside from higher myocardial vulnerability, diabetes mellitus may be associated with the triggering of adaptive processes leading to paradoxically lower susceptibility to ischemia. It has been proposed that this phenomenon shares some molecular pathways with short-term preconditioning and other forms of endogenous protection against ischemia/reperfusion injury in the nondiseased heart. The present article reviews some controversial findings of enhanced resistance to ischemia in the diabetic heart that stem from experimental studies in different models of myocardial ischemia/reperfusion injury. Specifically, it addresses the issue of potential mechanisms of increased resistance to ischemia in an experimental model of streptozotocin-induced diabetes, particularly with respect to the role of reactive oxygen species, hyperglycemia as one of the stress factors, and cell-signalling mechanisms mediated by ‘prosurvival’ cascades of protein kinases in relation to the mechanisms of classical ischemic preconditioning. Finally, mechanisms involved in the suppression of protection in the diabetic myocardium including the effect of concomitant pathology, such as hypercholesterolemia, are discussed.
PMCID: PMC3016064  PMID: 21264077
Adaptation; Cell signalling; Diabetic heart; Myocardial ischemia
18.  Chemokine expression during hepatic ischemia/reperfusion-induced lung injury in the rat. The role of epithelial neutrophil activating protein. 
Journal of Clinical Investigation  1995;95(1):134-141.
The liver is highly susceptible to a number of pathological insults, including ischemia/reperfusion injury. One of the striking consequences of liver injury is the associated pulmonary dysfunction that may be related to the release of hepatic-derived cytokines. We have previously employed an animal model of hepatic ischemia/reperfusion injury, and demonstrated that this injury causes the production and release of hepatic-derived TNF, which mediates a neutrophil-dependent pulmonary microvascular injury. In this study, we have extended these previous observations to assess whether an interrelationship between TNF and the neutrophil chemoattractant/activating factor, epithelial neutrophil activating protein-78 (ENA-78), exists that may be accountable for the pathology of lung injury found in this model. In the context of hepatic ischemia/reperfusion injury, we demonstrated the following alterations in lung pathophysiology: (a) an increase in pulmonary microvascular permeability, lung neutrophil sequestration, and production of pulmonary-derived ENA-78; (b) passive immunization with neutralizing TNF antiserum resulted in a significant suppression of pulmonary-derived ENA-78; and (c) passive immunization with neutralizing ENA-78 antiserum resulted in a significant attenuation of pulmonary neutrophil sequestration and microvascular permeability similar to our previous studies with anti-TNF. These findings support the notion that pulmonary ENA-78 produced in response to hepatic-derived TNF is an important mediator of lung injury.
PMCID: PMC295389  PMID: 7814607
19.  Enhanced Fibrinolysis Protects Against Lung Ischemia-Reperfusion Injury 
Ischemia-reperfusion injury (IRI) continues to plague the field of lung transplantation resulting in suboptimal outcomes. In acute lung injury processes such as ventilator-induced injury, sepsis, or acute respiratory distress syndrome, extravascular fibrin has been shown to promote lung dysfunction and the acute inflammatory response. This study investigates the role of the fibrinolytic cascade in lung IRI and investigates the interplay between the fibrinolytic system and the inflammatory response.
Mice lacking the plasminogen activator inhibitor-1 gene (PAI-1 knock out, PAI-1 KO; and thus increased lysis of endogenous fibrin) and wild-type mice underwent in-situ left lung ischemia and reperfusion. Fibrin content in the lung was evaluated by immunoblotting. Reperfusion injury was assessed by histology and physiologic parameters. Proinflammatory mediators were measured in bronchoalveolar lavage fluid and plasma using enzyme-linked immunosorbent assays.
Ischemia-reperfusion causes fibrin deposition in murine lungs. Less fibrin was seen in PAI-1 KO mice compared to wild-type mice subjected to the same ischemia-reperfusion conditions. By histologic criteria, more evidence of IRI was noted (thickening of the interstium, cellular infiltration in the alveoli) in the wild type than in PAI-1 KO mice. Physiologic parameters also revealed more IRI in the wild-type compared to PAI-1 KO mice. Cytokine and chemokines were elevated more in the wild-type group than the PAI-1 KO group.
Lung IRI triggers fibrin deposition in the murine lungs and fibrin creates a proinflammatory environment. Preventing fibrin deposition may reduce IRI and inflammation. This finding may lead to novel treatment strategies for ischemia-reperfusion.
PMCID: PMC2752840  PMID: 19379998
ischemia-reperfusion injury; lung transplantation; lung injury
20.  The role of 17-beta estradiol in ischemic preconditioning protection of the heart 
The protective effects of 17-beta estradiol (E2) on cardiac tissue during ischemia/reperfusion (I/R) injury have not yet been fully elucidated.
To assess the protective effects of short- and long-term E2 treatments on cardiac tissue exposed to I/R, and to assess the effects of these treatments in combination with ischemic preconditioning (IPC) on cardiac protection from I/R injury.
Sprague Dawley rats were assigned to the following treatment protocols: control (no preconditioning); IPC (isolated hearts were subjected to two cycles of 5 min global ischemia followed by 10 min of reperfusion); E2 preconditioning (E2PC; isolated hearts were subjected to E2 pharmacological perfusion for 15 min); short-term in vivo E2 pretreatment for 3 h; long-term in vivo E2 pretreatment or withdrawal (ovariectomy followed by a six-week treatment with E2 or a placebo); combined IPC and E2PC; combined IPC and short- or long-term E2 pretreatments or withdrawal. All hearts were isolated and stabilized for at least 30 min before being subjected to 40 min of global ischemia followed by 30 min of reperfusion; left ventricular function and vascular hemodynamics were then assessed.
IPC, E2PC and short-term E2 pretreatment led to the recovery of left ventricle function and vascular hemodynamics. Long-term E2 and placebo treatments did not result in any protection compared with untreated controls. The combination of E2PC or short-term E2 treatments with IPC did not block the IPC protection or result in any additional protection to the heart. Long-term E2 treatment blocked IPC protection; however, placebo treatment did not.
Short-term treatment with E2 protected the heart against I/R injury through a pathway involving the regulation of tumour necrosis factor-alpha. The combination of short-term E2 treatment with IPC did not provide additional protection to the heart. Short-term E2 treatment may be a suitable alternative for classical estrogen replacement therapy.
PMCID: PMC3628420  PMID: 23620695
Estrogen; Ischemia; Preconditioning; Reperfusion; Tumour necrosis factor-alpha
21.  Comparison of lung preservation solutions in human lungs using an ex vivo lung perfusion experimental model 
Clinics  2012;67(9):1101-1106.
Experimental studies on lung preservation have always been performed using animal models. We present ex vivo lung perfusion as a new model for the study of lung preservation. Using human lungs instead of animal models may bring the results of experimental studies closer to what could be expected in clinical practice.
Brain-dead donors whose lungs had been declined by transplantation teams were used. The cases were randomized into two groups. In Group 1, Perfadex® was used for pulmonary preservation, and in Group 2, LPDnac, a solution manufactured in Brazil, was used. An ex vivo lung perfusion system was used, and the lungs were ventilated and perfused after 10 hours of cold ischemia. The extent of ischemic-reperfusion injury was measured using functional and histological parameters.
After reperfusion, the mean oxygenation capacity was 405.3 mmHg in Group 1 and 406.0 mmHg in Group 2 (p = 0.98). The mean pulmonary vascular resistance values were 697.6 and 378.3 dyn·s·cm-5, respectively (p = 0.035). The mean pulmonary compliance was 46.8 cm H2O in Group 1 and 49.3 ml/cm H2O in Group 2 (p = 0.816). The mean wet/dry weight ratios were 2.06 and 2.02, respectively (p = 0.87). The mean Lung Injury Scores for the biopsy performed after reperfusion were 4.37 and 4.37 in Groups 1 and 2, respectively (p = 1.0), and the apoptotic cell counts were 118.75/mm2 and 137.50/mm2, respectively (p = 0.71).
The locally produced preservation solution proved to be as good as Perfadex®. The clinical use of LPDnac may reduce costs in our centers. Therefore, it is important to develop new models to study lung preservation.
PMCID: PMC3438253  PMID: 23018310
Lung Transplantation; Organ Preservation; Ischemia-Reperfusion Injury
22.  Intraoperative Hyperglycemia Augments Ischemia Reperfusion Injury in Renal Transplantation: A Prospective Study 
Journal of Transplantation  2011;2011:652458.
Background. Diabetes is a risk factor for delayed graft function in kidney transplantation, and hyperglycemia increases ischemia reperfusion injury in animal models. Methods. To explore the role of perioperative hyperglycemia in ischemia reperfusion injury, we conducted a prospective study of 40 patients undergoing living donor renal transplantation. Blood glucose levels were monitored intraoperatively, and serum samples were obtained at the time anesthesia was induced and one hour after allograft reperfusion. The percentage change in neutrophil gelatinase-associated lipocalin (NGAL), a protein whose expression is increased with renal ischemia, was then used to determine the extent of injury. Results. In a multivariate model including recipient, donor, and transplant factors, recipient blood glucose >160 mg/dL at the time of allograft reperfusion (β 0.19, P-value < 0.01), warm ischemia time >30 minutes (β 0.11, P-value 0.13), and recipient age (β 0.05, P-value 0.05) were associated with percentage change in NGAL. These same predictors were associated with the percentage change in creatinine on postoperative day 2. Conclusions. Hyperglycemia is associated with increased ischemic injury in renal transplantation. Both creatinine and NGAL, a marker of ischemic injury and renal function, fall less rapidly in patients with elevated blood glucose.
PMCID: PMC3166717  PMID: 21904663
23.  Small GTPases in mechanosensitive regulation of endothelial barrier 
Microvascular research  2008;77(1):46-52.
Alterations in vascular permeability are defining feature of diverse processes including atherosclerosis, inflammation, ischemia/reperfusion injury, and ventilator-induced lung injury. Clinical observations and experimental studies support an essential role of mechanical forces in pathophysiologic regulation of lung barrier. Accumulating data demonstrate that decreased levels of blood flow and increased cyclic stretch of lung tissues associated with lung mechanical ventilation at high tidal volumes increases vascular permeability, activates inflammatory cytokine production, alveolar flooding, leukocyte infiltration, hypoxemia, and increases morbidity and mortality. Potential synergism between pathologic mechanical stimulation and inflammatory molecules resulting in vascular leak and lung injury becomes increasingly recognized. This review will discuss a role of Rho family of small GTPases in the mechanochemical regulation of pulmonary endothelial permeability associated with ventilator induced lung injury.
PMCID: PMC2673191  PMID: 18938185
24.  Influence of Ketotifen, Cromolyn Sodium, and Compound 48/80 on the survival rates after intestinal ischemia reperfusion injury in rats 
BMC Gastroenterology  2008;8:42.
Mast cells were associated with intestinal ischemia-reperfusion injury, the study was to observe the influence of Ketotifen, Cromolyn Sdium(CS), and Compound 48/80(CP) on the survival rates on the third day after intestinal ischemia-reperfusion injury in rats.
120 healthy Sprague-Dawley rats were randomly divided into 5 groups, Sham-operated group (group S), model group (group M), group K, group C and group CP. Intestinal damage was triggered by clamping the superior mesenteric artery for 75 minutes, group K, C, and CP were treated with kotifen 1 mg·kg-1, CS 50 mg·kg-1, and CP 0.75 mg·kg-1 i.v. at 5 min before reperfusion and once daily for three days following reperfusion respectively. Survival rate in each group was recorded during the three days after reperfusion. All the surviving rats were killed for determining the concentration of serum glutamic-oxaloacetic transaminase(AST), glutamic pyruvic transaminase(ALT), the ratio of AST compare ALT(S/L), total protein(TP), albumin(ALB), globulin(GLB), the ratio of ALB compare GLB(A/G), phosphocreatine kinase(CK), lactate dehydrogenase(LDH), urea nitrogen(BUN) and creatinine(CRE) at the 3rd day after reperfusion. And ultrastructure of IMMC, Chiu's score, lung histology, IMMC counts, the levels of TNF-α, IL-1β, IL-6 and IL-10 of the small intestine were detected at the same time.
Intestinal ischemia-reperfusion injury reduced the survival rate. The concentrations of TP, ALB and level of IL-10 in intestine in group M decreased significantly while the concentrations of S/L, LDH and the levels of IL-6 and TNF-α in intestine increased significantly compared with group S (P < 0.05). Treatment with Ketotifen and CS increased the survival rate compared with group M (P < 0.05), attenuated the down-regulation or up-regulation of the above index (P < 0.05). Treatment with CP decreased the survival rate on the 3rd day after reperfusion compared with group M(P < 0.05). Group K and C had better morphology in IMMC in the small intestine and in the lungs than in group M and CP, although the Chiu's score and IMMC counts remained the same in the five groups(P > 0.05).
Mast cell inhibition after ischemia prior to reperfusion and following reperfusion may decrease the multi-organ injury induced by intestine ischemia reperfusion, and increase the survival rates.
PMCID: PMC2564972  PMID: 18808687
25.  Delayed VEGF Treatment Enhances Angiogenesis and Recovery After Neonatal Focal Rodent Stroke 
Translational stroke research  2013;4(2):189-200.
Neonatal stroke occurs in one in 4,000 live births and leads to significant morbidity and mortality. Approximately two thirds of the survivors have long-term sequelae including seizures and neurological deficits. However, the pathophysiological mechanisms of recovery after neonatal stroke are not clearly understood, and preventive measures and treatments are nonexistent in the clinical setting. In this study, we investigated the effect of vascular endothelial growth factor (VEGF) treatment on histological recovery and angiogenic response to the developing brain after an ischemic insult. Ten-day-old Sprague–Dawley rats underwent right middle cerebral arterial occlusion (MCAO) for 1.5 h. Diffusion-weighted MRI during occlusion confirmed focal ischemia that was then followed by reperfusion. On group of animals received 5-bromo-2-deoxyuridine and sacrificed at postnatal day (P)18 or P25. A second group of animals was treated with VEGF (1.5 µg/kg, icv) or phosphate-buffered saline (PBS) at P18 and perfusion fixed at P25. Based on Nissl and iron staining, a single VEGF injection reduced the injury score, compared to the animals that underwent MCAO and PBS injection. Furthermore, neurodegeneration represented by neuronal nuclei staining was markedly diminished. In addition, animals treated with VEGF revealed a positive trend in endothelial proliferation and a significant increase in total vessel volume in the peri-infarct region of the caudate. The number of Iba1-positive microglial cells was significantly reduced after a single VEGF injection, and myelin basic protein expression was enhanced in the caudate after ischemia without an effect of VEGF treatment. In conclusion, delayed treatment with VEGF ameliorates injury, promotes endothelial cell proliferation, and increases total vascular volume following neonatal stroke. These results suggest that VEGF has a neuroprotective effect, in part by enhancing endogenous angiogenesis. These data contribute to a better understanding of neonatal stroke.
PMCID: PMC3733573  PMID: 23926451
VEGF; Neuroprotection; Angiogenesis; Neonatal stroke

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